Many normal and abnormal physical conditions, such as, menopause, aging, fractures and other diseases alter the state of bone remodeling. The osteogenic factor bone morphogenetic protein-2 (BMP-2) is highly expressed in the microenvironment of bone remodeling. BMP-2 is necessary for osteoblast growth, differentiation and survival to form mature bone. The mechanism by which BMP-2 is expressed in preosteoblasts and the underlying signal transduction pathways of osteoblast differentiation are being actively characterized. Our preliminary data provide the first evidence that BMP-2 increases phosphatidylinositol 3 kinase (PI 3 K)/Akt kinase signaling, which regulates osteoblast differentiation. Furthermore, we demonstrate that BMP-2 regulates its own expression by activating two transcription factors, NFKappaB and MEF-2A. Moreover, BMP-2 induces reactive oxygen species (ROS) in primary osteoblasts with concomitant increase in PI 3 K-dependent NADPH oxidase activity. Statins have recently been shown to induce new bone formation by inducing BMP-2 expression. Our preliminary data show that statin stimulates PI 3 K/Akt signaling in preosteoblasts. In this proposal, using preosteoblast cell line and primary fetal rat calvarial cells, we will test the hypothesis that concerted action of redox and PI 3 K/Akt signaling regulates BMP-2 expression via NFKappaB and MEF-2A to induce osteoblast differentiation. In the first specific aim, we plan to investigate the role of PI 3 K/Akt signaling cascade in the regulation of NFKappaB and MEF-2A transcription factors. In the second specific aim, we will examine the role of ROS, a downstream mediator of PI 3 K, in osteoblast differentiation in response to BMP-2. In the specific aim 3, we will study the PI 3 K/Akt signaling pathway as mechanism for statin-induced BMP-2 expression and osteoblast differentiation. To address these specific aims, techniques including immunoprecipitation, immunoblotting, immunecomplex kinase assays, electrophoretic mobility shift assay, reporter transfection assays, adenovirus-mediated gene transfer of mutant enzymes and conditional expression of proteins will be used. Understanding the signal transduction pathways of osteoblast differentiation may result in the development of therapeutic modalities for the human diseases where lack of bone formation is the pathology.